Homologous recombination refers to the exchange of homologous segments anywhere along a length of two DNA nucleotide sequences. In normal mitotic and meiotic cell cycle processes, homologous recombination may occur during spontaneous strand breaks of the DNA in the case of mitotic cells, or it may be catalysed by a specific enzyme such as SPO11 in the case of meiosis. Upon formation of the double-strand breaks, the exposed double-stranded ends are thought to be resected by an exonuclease activity that degrades the DNA to generate single-stranded DNA (ssDNA) ends which have a 3′ hydroxyl group. The single-stranded ends may then be acted upon by strand invasion proteins such as RecA homologues. Many of these proteins may be specific to mitotic or meiotic cells, whereas other proteins act in both mitotic and meiotic cells. Strand invasion, pairing of homologous nucleotide sequences and formation of initial crossovers (chiasmata) between the sister chromatids in vegetative cells and non-sister chromatids in meiotic cells is followed by branch migration, DNA replication and strand displacement. Resolution of the crossovers formed between paired chromatids may be enhanced by proteins known as resolvases which have been characterized in a variety of organisms.
Homologous recombination may be employed to genetically engineer DNA sequences in an organism and provides a basis for performing specific targeted alterations in the genome of an organism. Targeted homologous recombination has been proposed to create transgenic organisms such as plants and animals. A transgenic organism is one that contains a copy of a gene or nucleotide sequence from another species. Homologous recombination permits the targeted change to be incorporated into homologous sites in the chromosomes of the organism so that the change may be passed on to the organism's progeny.
There are a variety of references that disclose methods of modulating homologous recombination in an organism. For example, WO 02/22811 discloses methods of modifying the level or functional activity of factors such as enzymes or other catalytic proteins to modify the frequency of meiotic homologous recombination in a eukaryotic cell. The invention provides methods of increasing meiotic homologous recombination in a eukaryote by transforming a cell with an activator of homologous recombination or inhibiting meiotic homologous recombination by transforming a cell with an inhibitor of meiotic recombination. While the patent application discloses several methods for modulating homologous recombination, there is a need in the art for alternate methods of modulating homologous and homeologous recombination in an organism.
PCT applications WO 01/62945, WO 01/61012 and U.S. Pat. No. 6,146,894 to Nicolaides et al., disclose methods for generating hypermutable yeast, plants and mammalian cells, respectively. The references teach expressing a dominant negative allele of an endogenous mismatch repair gene in a cell to generate hypermutable cells and organisms. By introduction of the dominant negative alleles, new cell lines and organism varieties can be prepared more efficiently than by relying on the natural rate of mutation. Specifically, WO 01/61012 discloses transforming a cell or transgenic plant with a dominant negative allele encoding a protein which is part of a mismatch repair complex such as MutS or MutL homologues of the bacterial, yeast, fungal, insect, plant or mammalian genes.
Although the patents and applications suggest that the disclosed methods result in hypermutable cells, a major problem with such systems is that it may take an excessively long time for the hypermutable cells to recombine to give a specific desired mutation. Further there is no suggestion in any of the references of a method of increasing or decreasing homeologous recombination during inter or intraspecies breeding. Thus, there is a need in the art for novel methods of increasing or decreasing the recombinogenic potential of cells, for example during meiosis. There is a need in the art for novel methods of increasing or decreasing homeologous recombination during inter or intraspecies breeding. Further there is a need for alternate sources of mismatch repair proteins, for example, prokaryotic proteins including MutS, for use in in vivo mutagenesis
It is an object of the present invention to overcome disadvantages of the prior art.
The above object is met by a combination of the features of the main claims. The sub claims disclose further advantageous embodiments of the invention.